MicroRNAs (miRNAs) have been recognized as an important class of non-coding RNAs that are involved in a variety of biological and pathological processes. The broad, long-term objectives are to elucidate the molecular mechanism through which long miRNA primary transcripts (pri-miRNAs) are specifically recognized by cellular factors for processing and how their maturation is regulated through a mechanism involving heme. Specifically, we will focus on the structure and function of a key factor-an human RNA binding protein called DGCR8 that is heterozygously deleted in DiGeorge syndrome patients. We expect to fill major gaps in our current knowledge of miRNAs: some critical features of miRNA genes have not been identified so that miRNA genes cannot be accurately predicted from genomic sequences;and little is known about how the processing of miRNAs are regulated. Specific aim 1 will elucidate how DGCR8 interacts with pri-miRNAs. The protein-RNA interface will be examined using biochemical and X-ray crystallographic methods. The origin of the binding cooperativity will be investigated using mutagenesis methods. In specific aim 2, the interactions between DGCR8 and the heme cofactor will be revealed. The amino acid residues important for heme binding will be identified using site-directed mutagenesis and screening methods. The structure of the heme-binding domain will be determined using X-ray crystallography or NMR. The molecular mechanism of autoinhibition of DGCR8 will be examined using in vitro methods. In specific aim 3, the importance of the heme-DGCR8 interaction and the autoinhibition of DGCR8 in pri-miRNA processing will be investigated in human cell cultures. Medical revelence: MicroRNAs are a new class of genes that are involved in important biological and disease processes. Our research aims to improve our abilities to find these genes in the human genome, to develop better theraprutic microRNAs that can modulate abnormal gene expression in disease states. Our discovery that heme may be involved in regulating the processing of microRNAs suggest that they can be used as therapeutic agents for microRNA-related diseases.